ES cells can proliferate without limit, and even after prolonged culture, retain the ability to form cells ranging from cardiac muscle to nerve to blood; potentially any cell type that makes up the body. The recent derivation of nonhuman primate (NHP), i.e. rhesus monkey and common marmoset, and human ES cells has widespread implications for human developmental biology, drug discovery, drug testing, and transplantation medicine. Because of the close embryological similarities between humans and old world monkeys, rhesus monkey ES cells and rhesus monkeys provide an extremely accurate, necessary model system for developing human ES cell-based therapies. The efficient genetic manipulation of primate ES cells is essential to: 1) elucidate gene function both during differentiation and in specific differentiated cells; 2) direct the differentiation of ES cells to specific lineages by the manipulation of transcription factors; 3) purify desired differentiated cell types from a mixed population of ES cell derivatives by introducing selectable markers; 4) use the differentiated derivatives of primate ES cells as vehicles for gene therapy; and 5) modulate the immune response to transplanted ES cell derivatives. Unfortunately, primate ES cells are extremely difficult to transfect. Transfection methods routinely used for mouse ES cells fail for primate ES cells. Recently, a series of pseudotyped, self-inactivating lentiviral vectors with internal promoters were tested. These are the first vectors tested that allow the derivation of rhesus and marmoset ES cell lines stably expressing a foreign gene. To improve these vectors for primate ES cells, and to use them as tools to dissect mechanisms of primate ES cell self-renewal, the following Specific Aims will be accomplished to: 1) generate lentiviral vectors with improved long term, site independent expression in primate ES cells and their differentiated derivatives by testing the effects of insulator sequences and scaffold attachment regions; 2) use a functional ES cell screen to isolate novel genomic DNA fragments that promote long term, integration site independent expression of lentiviral vectors; 3) use lentiviral vectors to test the role of specific genes involved in the self-renewal of other stem cells (Beta-catenin, ID-1, N-myc, Notch, STAT-3) in promoting the self-renewal of primate ES cells; and 4) use a lentiviral cDNA expression cloning strategy to identify novel genes produced by fibroblasts, or by the ES cells themselves that promote ES cell self-renewal.